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Carboxy SNARF-1 nanoreactors protected from dye-protein interactions, with fast pH response and reduced photobleaching for use in biological fluids.


Development of an environment independent dual-wavelength fluorescent nano-pH sensor is presented. The sensor is made of nanosized calcium and phosphate composite shell covering phosphatidylcholine liposomes and carboxy-SNARF-1 (cSNARF-1) dye. Particles were viewed with an electron microscope and their size dispersion analyzed with dynamic light scattering estimating the mean size to be 150 nm. Comparative analyses of two-wavelength fluorescence pH titration with and without the shell coating of the dye were carried out in solution, and in the presence of the plasma, albumin and IGG. The results demonstrated that serious distortions of the pH titration plots in the presence of albumin and plasma were minimized in the shell-coated dye and the results analyzed with the modified Henderson-Hasselbach equation to estimate the pH values of the solutions. Elucidated pH values were stable over time, photobleaching resistant, and reproducible under cyclic pH changes. Stopped flow fluorometric analysis revealed the pH response time was less than 200 msec and fluorescence microscopic observation was able to detect the particles and their colorimetric response in reference to the pH change, while maintaining the integrity of the particles under continuous illumination up to 80 min. Knowing that the number of the dye molecules in each particle is about 10, the measurement of pH can be achieved quickly with only small number of protons in the nanoreactor, making this protected and extended pH range sensor ideal for use in biological cells, microfluidics and other complex nano environments.